SPOP inhibits BRAF-dependent tumorigenesis through promoting non-degradative ubiquitination of BRAF

Background The gene encoding the E3 ubiquitin ligase substrate-binding adapter Speckle-type BTB/POZ protein (SPOP) is frequently mutated in prostate cancer (PCa) and endometrial cancer (EC); however, the molecular mechanisms underlying the contribution of SPOP mutations to tumorigenesis remain poorly understood. Methods BRAF harbors a potential SPOP-binding consensus motif (SBC) motif. Co-immunoprecipitation assays demonstrated that BRAF interacts with SPOP. A series of functional analyses in cell lines were performed to investigate the biological significance of MAPK/ERK activation caused by SPOP mutations. Results Cytoplasmic SPOP binds to and induces non-degradative ubiquitination of BRAF, thereby reducing the interaction between BRAF and other core components of the MAPK/ERK pathway. SPOP ablation increased MAPK/ERK activation. EC- or PCa-associated SPOP mutants showed a reduced capacity to bind and ubiquitinate BRAF. Moreover, cancer-associated BRAF mutations disrupted the BRAF-SPOP interaction and allowed BRAF to evade SPOP-mediated ubiquitination, thereby upregulating MAPK/ERK signaling and enhancing the neoplastic phenotypes of cancer cells. Conclusions Our findings provide new insights into the molecular link between SPOP mutation-driven tumorigenesis and aberrant BRAF-dependent activation of the MAPK/ERK pathway. Supplementary Information The online version contains supplementary material available at 10.1186/s13578-022-00950-z.


Introduction
The MAPK/ERK pathway, also known as the RAS-RAF-MEK-ERK pathway, transmits extracellular proliferative signals from the ligand-mediated activation of receptor tyrosine kinases to the cell nucleus [1,2]. BRAF is a member of the rapidly accelerated fibrosarcoma (RAF) kinase family, which includes ARAF and CRAF (RAF1) [3]. RAFs transduce signals downstream of the RAS via the MEK-ERK cascade. Among the three RAF kinases, BRAF binds most avidly to RAS and is the most active in phosphorylating MEK1/2 [4]. Oncogenic BRAF mutations have been detected in approximately 6% of human cancers, with most occurring in hairy cell leukemia (> 97%), melanoma (40-50%), thyroid cancer (30-50%), colorectal cancer (10%), and non-small cell lung cancer (3-5%) [5]. In contrast, mutations in ARAF and CRAF are relatively rare in human cancers. Oncogenic BRAF mutations lead to the constitutive activation of the MAPK/ERK pathway, resulting in uncontrolled proliferation, survival, invasiveness, and drug resistance [1]. BRAF inhibitors such as vemurafenib, dabrafenib, and encorafenib are currently used to treat patients with BRAF-mutant cancers [6,7].
Cullin-RING E3 ubiquitin ligases (CRLs) are a superfamily of modular, multi-subunit E3 ubiquitin ligases that mediate the ubiquitination and degradation of various cellular proteins involved in a plethora of physiological and pathological processes. Human cells express seven different Cullins (CUL1, 2, 3, 4A, 4B, 5, and 7), each of which nucleates a multi-subunit complex [8]. The Cullin3-RING E3 ubiquitin ligase complex subfamily employs a BTB domain-containing protein for substratebinding. Speckle-type BTB/POZ protein (SPOP) is a substrate-binding adaptor of CRL3s [9]. Investigation of the pathophysiological functions of SPOP has attracted extensive attention in recent years because of its high mutation frequency in prostate cancer (PCa) (5-15%) and endometrial cancer (EC) (8-10%), two hormonerelated cancers [10,11]. Recurrent SPOP mutations have also been detected in other cancer types, although at a relatively low frequency. SPOP mutations occur as heterozygous missense mutations with dominant-negative, selective loss-of-function effects on the remaining wildtype allele [12]. SPOP selectively recruits substrates via its N-terminal MATH domain, whereas its BTB and BACK domains mediate oligomerization and interaction with CUL3 [9]. The vast majority of EC-or PCa-associated SPOP mutations occur in the substrate-binding MATH domain. To date, dozens of SPOP substrates (AR, ERα, SRC-3, BET, GLP, etc.) have been identified through different biochemical methods and functionally investigated in PCa or EC settings, and the list of SPOP substrates is still growing [13][14][15][16][17]. Research has shown that cancerassociated SPOP mutants generally display an impaired capacity to bind substrates, leading to reduced ubiquitination of substrates. Accumulating evidence from cancer cell lines and animal models has demonstrated that SPOP mutations promote the initiation and progression of EC and PCa, potentially owing to the dysregulation of SPOP-mediated ubiquitination of its substrates [15,18]. However, the cellular pathways affected by SPOP mutations are not yet fully understood.
In this study, we identified BRAF as a bona fide substrate of the CRL3-SPOP E3 ubiquitin ligase complex. The CRL3-SPOP complex mediates non-degradative ubiquitination of BRAF, which leads to attenuated MAPK/ERK activation. Moreover, mutations in the SPOP-BRAF-binding interface disrupt the SPOP-BRAF regulatory pattern and promote aberrant MAPK/ERK activation and malignant transformation in cancer cells.

Plasmid constructions
Expression vectors for SPOP-WT and its mutants have been described previously [14]. BRAF cDNA was obtained from Genecopia and subcloned into the pCMV vector. BRAF mutants were generated using the KOD-Plus-Mutagenesis Kit (TOYOBO) following the manufacturer's instructions.

CRISPR-Cas9 mediated gene knock out stable cell generation
The pX459 plasmid was used to clone guide oligonucleotides targeting BRAF and SPOP genes. Ishikawa and SPEC-2 cells were plated and transfected overnight with pX459. 24 h after transfection, 1 μg/ml puromycin was used to screen cells for 3 days. Living cells were seeded in 96 well plate at a limited dilution to isolate the monoclonal cell lines. KO cell clones were screened by Western blot (WB) and validated by Sanger sequencing. The sequences of gene-specific sgRNAs are listed in Additional file 1: Table S1.

Protein half-life assays
To measure protein half-life, cycloheximide (50 μg/ml) was added to the media of the parental and SPOP-KO Ishikawa cells. At the indicated time points, cell lysates were prepared and analyzed by WB using the indicated antibodies.

Colony formation assays
Ishikawa cells (1 × 10 3 ) were seeded in triplicate in 6-well plates containing each well. After incubation for 2 weeks, the cells were fixed with 4% paraformaldehyde for 15 min and stained with Giemsa dye (Solarbio) for 20 min. The cells were then washed with water by gentle dropping and air dried at room temperature. The number of colonies was quantified using a Nikon digital camera and the ImageJ software.
In vivo ubiquitination assays 293 T cells were transfected with HA-Ubiquitin and other indicated constructs. 36 h after transfection, cells were lysed in 1% SDS buffer (Tris [pH 7.5], 0.5 mM EDTA, 1 mM DTT) and boiled for 10 min. For co-immunoprecipitation (Co-IP), cell lysates were diluted tenfold in Tris-HCl buffer and incubated with anti-FLAG M2 agarose beads (Sigma) for 4 h at 4 °C. The bound beads were then washed four times with BC100 buffer (20 mM Tris-Cl, pH 7.9, 100 mM NaCl, 0.2 mM EDTA, 20% glycerol) containing 0.2% Triton X-100. Proteins were eluted with 3X FLAG peptide for 2 h at 4 °C. The ubiquitinated form of BRAF was detected by WB with anti-HA antibody.

Cell proliferation assays
The proliferation rate of Ishikawa cells was determined using the Cell Counting Kit-8 (CCK-8) Kit (Beyotime) according to the manufacturer's instructions. Briefly, the cells were seeded onto 96-well plates at a density of 1 × 10 3 cells/well in triplicate. During the 2-to 6 day culture period, 10 μl of CCK-8 solution was added to the cell culture and incubated for 2 h. The absorbance of the samples was measured at 450 nm wavelength using a microplate absorbance reader.

Migration and invasion assays
Cell migration and invasion were determined using Transwell (Costar) migration and invasion assays. Ishikawa cells were precultured in serum-free medium for 48 h. For the migration assay, 3 × 10 4 cells were seeded in serum-free medium in the upper chamber, and the lower chamber was filled with DMEM containing 5% FBS. After 48 h, the non-migrating cells in the upper chambers were carefully removed with a cotton swab and migrated cells underside of the filter were stained and counted in nine different fields. Matrigel invasion assays were performed using Transwell inserts (Costar) coated with Matrigel/ fibronectin (BD Biosciences).

Sphere formation assays
Ishikawa cells (1 × 10 3 cells/well) were mixed with Matrigel (BD Biosciences) and plated in 24-well ultralow-attachment plates (Corning) in DMEM containing 10% FBS. Fresh medium was added every 3 days. Floating spheres that grew in 1-2 weeks were quantified using a Nikon digital photo camera and the ImageJ software.

Immunofluorescence and confocal microscopy
For immunofluorescence, cells were plated on chamber slides and fixed with 4% paraformaldehyde at room temperature for 30 min. After washing with PBS, cells were permeabilized with 0.1% Triton X-100 in PBS for 15 min. The cells were then washed with PBS, blocked with 0.5% BSA in PBS for 1 h, and incubated with primary antibodies in PBS at 4 °C overnight. After washing with PBS, fluorescence-labeled secondary antibodies were applied and DAPI was counterstained for 1 h at room temperature. The cells were visualized and imaged using a confocal microscope (LSM710, Zeiss).

Statistical analysis
Statistical analysis was performed using GraphPad Prism (GraphPad Software, Inc.), and all data are shown as mean values ± standard deviation (SD) for experiments performed with at least three replicates. The differences between the two groups were analyzed using one-way or two-way ANOVA test unless otherwise specified. * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001, **** represents p < 0.0001, n.s. represents non-significant.

Identification of BRAF as a novel SPOP interactor
Previously, we and others have identified multiple oncoproteins as degradative or non-degradative substrates of the CRL-SPOP complex using the yeast two-hybrid method or affinity purification coupled with mass spectrometry. Substrate-binding consensus (SBC) motifs (Φ-π-S/T-S/T-S/T, where Φ is a nonpolar residue and π is a polar residue) have been well-characterized in the majority, if not all, of these substrates [9]. To expand the SPOP interactome to a proteome-wide level, we performed an SBC motif search against the human proteome sequence using the webtool ScanProsite (https:// prosi te. expasy. org/ scanp rosite/). Among the hundreds of potential candidates identified by this search, we noticed that BRAF harbors a perfectly matched SBC motif (120-VTSSS-124 aa), which is very similar to those present in several known SPOP substrates, including ERG, ATF2, Caprin1, and DAXX (Fig. 1a). Notably, this motif was absent in BRAF paralogs of ARAF and CRAF (Fig. 1b). Given that BRAF is frequently hyperactivated in various human cancers, we explored whether BRAF is an authentic substrate of the CRL3-SPOP complex, and whether BRAF activity is dysregulated in SPOP-mutated cancers. Using reciprocal co-IP assays, we demonstrated that ectopically expressed BRAF interacted with SPOP ( Fig. 1c, d). In contrast, ectopically expressed ARAF or CRAF did not interact with SPOP (Fig. 1e). Only SPOP, but none of the other CRL3-based BTB domain-containing adaptors examined, interacted with BRAF (Fig. 1f ). Moreover, a specific endogenous interaction between SPOP and BRAF was observed in Ishikawa endometrial cancer cells (Fig. 1g, h). In accordance with a previous study showing that the MATH domain of SPOP is responsible for recruiting substrates [9], deletion of the MATH domain, but not the CUL3-binding BTB domain, completely abolished the interaction between SPOP and BRAF (Fig. 1i, j). Taken together, our data indicate that SPOP interacts with BRAF in cells.

SPOP promotes non-degradative ubiquitination of BRAF
Next, we investigated whether BRAF protein stability was controlled by the ubiquitin-proteasome pathway. Treatment of Ishikawa cells with the proteasome inhibitor MG132 had no obvious effect on BRAF protein levels, indicating that BRAF has a relative long half-life, at least in Ishikawa cells. MLN4924, a small-molecule inhibitor of the NEDD8-activating enzyme required for the activation of the CRL complex, did not alter BRAF protein levels. In contrast, MG132 or MLN4924 treatment caused a marked increase in the protein levels of BRD4 and Caprin1, which are two reported substrates of SPOP (Fig. 2a). Ectopic overexpression of SPOP-WT or its domain deletion mutants did not alter the levels of co-expressed BRAF (Fig. 2b). To characterize the effect of SPOP on endogenous BRAF, we generated Tet-oninducible Ishikawa cells. Induction of FLAG-SPOP by doxycycline treatment did not affect BRAF protein levels, whereas BRD4 was destabilized in a time-dependent manner (Fig. 2c). Depletion of SPOP by shRNA-mediated knockdown or CRISPR/Cas9-mediated KO in EC cell lines elevated the protein levels of BRD4 and Caprin1, but not BRAF (Fig. 2d, Additional file 1: Fig. S1a-c). Moreover, the half-life of BRAF was comparable between parental and SPOP KO cells (Fig. 2e, f ). Although BRAF was not degraded by SPOP, it was robustly polyubiquitinated in response to the co-expression of SPOP-WT but not by the SPOP-ΔBTB or -ΔMATH mutant (Fig. 2g). We further demonstrated that the SPOP-CUL3-RBX1 E3 ubiquitin ligase complex catalyzed BRAF polyubiquitination in vitro (Additional file 1: Fig. S1d). Accordingly, SPOP depletion decreased the ubiquitination levels of endogenous BRAF (Fig. 2h). These results indicate that SPOP induces non-degradative polyubiquitination of BRAF. Given that SPOP-mediated BRAF ubiquitination is non-degradative, we examined the polyUb chain-linkage specificity of BRAF. We performed in vivo ubiquitination assays using a panel of ubiquitin mutants containing single KR mutations at each of the seven lysine residues. Co-expression of either Ub-KR mutant did not obviously alter SPOP-mediated BRAF ubiquitination. We also used a reciprocal series of Ub-KO mutants that contain only one lysine residue, with the other six lysine residues mutated to arginine. The expression of either Ub-KO mutant nearly abolished SPOP-mediated BRAF ubiquitination (Fig. 2i), indicating that SPOP may catalyze the synthesis of mixed-linkage polyUb chains on BRAF. We also utilized linkage-specific K27/K29/48/63-Ub antibodies to demonstrate that ubiquitinated BRAF contains K27-, K29-, K48-, and K63-Ub linkages (Additional file 1: Fig. S1e). Taken together, our data indicate that SPOP promotes non-degradative ubiquitination of BRAF.

The SBC motif in BRAF is required for SPOP-mediated BRAF ubiquitination
To examine whether the potential SBC motif is required for the SPOP-BRAF interaction, we generated a BRAF mutant in which the SBC motif was deleted. Co-IP assay results showed that SPOP only interacted with wildtype BRAF but did not bind to the BRAF-ΔSBC mutant (Fig. 3a, Additional file 1: Fig. S2a). The deletion of the SBC motif in BRAF abrogated SPOP-mediated BRAF ubiquitination (Fig. 3b). Mutations in amino acids in the SBC motif considerably reduced the SPOP-BRAF interaction (Fig. 3c) and SPOP-mediated BRAF ubiquitination (Fig. 3d).
Given that BRAF interacts with SPOP in an SBC motif-dependent manner, we further explored whether cancer-associated BRAF mutations that occur in the SBC motif would attenuate the SPOP-BRAF interaction and permit BRAF to evade SPOP-mediated ubiquitination. To this end, we examined the cancer sequencing data deposited in cancer gene mutation databases (cBioPortal and COSMIC). We identified a BRAF-T121I mutation in glioblastoma and BRAF-S122Y mutation in osteosarcoma (Fig. 1b, Additional file 1: Table. S3). We found that the SPOP-binding capacity of these BRAF mutants was markedly reduced (Fig. 3e, Additional file 1: Fig. S2b), and SPOP-mediated ubiquitination of these mutants was also markedly attenuated (Fig. 3f ). Taken together, our data indicate that cancer-derived BRAF mutations occurring in the SBC motif allow BRAF to evade SPOP-mediated ubiquitination.

EC-and PCa-associated SPOP mutants are defective in promoting BRAF ubiquitination
To date, the vast majority of SPOP mutations associated with EC or PCa occur within the MATH domain, which is responsible for selective substrate binding (Fig. 4a, b) [10,11]. We postulated that EC-or PCa-associated SPOP mutants may be defective in mediating BRAF ubiquitination. The BRAF-binding ability of EC-associated SPOP mutants was moderately or severely impaired as compared to that of SPOP-WT (Fig. 4c). SPOP-mediated BRAF ubiquitination was moderately or severely attenuated in these mutants (Fig. 4d). Similar effects were observed when PCa-associated SPOP mutants were tested (Fig. 4e, f ). In accordance with a previous study showing that cancer-associated SPOP mutants function as dominant-negative variants to deregulate their substrates [12], we found that co-expression of the SPOP mutant markedly reduced the interaction between SPOP-WT and BRAF (Fig. 4g), resulting in the suppression of wild-type SPOP-mediated BRAF ubiquitination (Fig. 4h). Taken together, our data indicate that EC or PCa-associated SPOP mutants exert dominant-negative effects by downregulating BRAF ubiquitination due to impaired BRAF binding capacity.

Cytoplasmic but not nuclear SPOP promotes BRAF ubiquitination
BRAF is a cytoplasmic protein kinase. Our previous study showed that SPOP is localized exclusively in the nucleus as speckles or in both the cytoplasm and nucleus [19]. We investigated the subcellular localization of the SPOP-BRAF interaction. BRAF was diffusely localized in the cytoplasm. When SPOP was localized in both the cytoplasm and nucleus, BRAF was recruited into SPOP speckles in the cytoplasm. When SPOP was localized exclusively in the nucleus, it did not co-localize with cytoplasmic BRAF. We found that SPOP lacking the NLS sequence (SPOP-ΔNLS) accumulated exclusively in the cytoplasm in punctate patterns and colocalized perfectly with cytoplasmic BRAF. Moreover, the deletion of the SBC motif in BRAF did not alter its diffuse cytoplasmic localization, and the SPOP-induced speckle pattern of BRAF was not observed (Fig. 5a).
Next, we investigated the effect of cancer-associated SPOP mutations on BRAF localization. We focused on three hotspot mutants (E47K, S80R, and W131G). Three cancer-associated SPOP mutants lacking the NLS sequence (ΔNLS-E47K, -S80R, and -W131G) accumulated exclusively in the cytoplasm in punctate patterns similar to those of SPOP-ΔNLS, but these mutants did not colocalize with BRAF. Similarly, the cancer-associated BRAF-T121I mutant did not co-localize with SPOP (Fig. 5b). Our previous studies showed that the SPOP-ΔNLS mutant was more effective in promoting the ubiquitination of its cytoplasmic substrates (INF2, MyD88, Caprin1, and HIPK2) than SPOP-WT, but it was unable to ubiquitinate nuclear substrates [19][20][21][22]. Indeed, we found that SPOP-ΔNLS immunoprecipitated more BRAF than SPOP-WT (Fig. 5c) and SPOP-ΔNLS was more effective in promoting BRAF ubiquitination than SPOP-WT (Fig. 5d). Taken together, our data indicate that SPOP can recruit BRAF into cytoplasmic speckles and that this activity strictly depends on its cytoplasmic localization.

SPOP suppresses MAPK/ERK pathway activation
BRAF plays a critical role in the regulation of MAPK/ ERK pathway activation [3]. We examined MAPK/ERK activity in SPOP KO and SPOP overexpressed cells. The induction of FLAG-SPOP by doxycycline treatment led to a marked decrease in the phosphorylation levels of MEK1/2, ERK1/2, and MSK1, indicating that MAPK/ ERK activation was attenuated (Fig. 6a). Conversely, stronger MAPK/ERK activation was observed when SPOP was ablated (Fig. 6b). Moreover, SPOP-ΔNLS overexpression attenuated the MAPK/ERK pathway activation in EGF-stimulated cells (Fig. 6c, d). Conversely, SPOP KO enhanced MAPK/ERK pathway activation in EGF-stimulated cells (Fig. 6e, f ). Stronger MAPK/ ERK activation was observed in SPOP KO Ishikawa cells reconstituted with the SPOP-S80R mutant than in those reconstituted with the SPOP-Y87C mutant, which retains a similar binding capacity to BRAF as SPOP-WT (Additional file 1: Fig. S3a, b).
To determine whether the escape of SPOP-mediated-BRAF ubiquitination has any impact on MAPK/ ERK pathway activation, we reconstituted BRAF-KO Ishikawa cells (Additional file 1: Fig. S3c-e) with BRAF-WT or the -T121I mutant, which is incapable of binding to SPOP. As shown in Fig. 6g, h, EGF-induced MAPK/ERK activation was stronger in BRAF-KO Ishikawa cells reconstituted with the BRAF-T121 mutant than in those reconstituted with BRAF-WT. We found that stable overexpression of SPOP-WT or ΔNLS mutant reduced the interaction between BRAF and its upstream regulator, H-RAS, and downstream effectors MEK1/2 and ERK1/2 (Fig. 6i). Conversely, stable overexpression of SPOP-S80R or -F125V, increased the interactions between BRAF and MAPK/ERK pathway components. Surprisingly, stable overexpression of the SPOP-Y87C mutant even moderately reduced the interactions between BRAF and MAPK/ERK pathway components (Fig. 6j). Moreover, the interactions between BRAF and MAPK/ERK pathway components were moderately increased in BRAF-KO Ishikawa cells reconstituted with the BRAF-T121 mutant as compared to those reconstituted with BRAF-WT (Fig. 6k). Taken together, our data indicate that SPOP suppresses MAPK/ERK pathway activation.

BRAF-T121I mutant is more effective to promote cell growth, migration, and invasion than BRAF-WT
We found that stable overexpression of SPOP-WT reduced the growth of Ishikawa, KLE, and SPEC-2 cells as compared to parental cells. In contrast, stable overexpression of EC-associated SPOP mutants increased the growth of KLE, SPEC-2, and Ishikawa cells as compared to parental cells (Additional file 1: Fig.  S4a-d). Consistent with the substantial roles of BRAF in cellular malignancy [5], BRAF KO Ishikawa cells grew at a markedly reduced growth rate, as demonstrated by colony formation and CCK-8 assays. Reconstitution with BRAF-WT restored BRAF-KO cell growth in vitro, and more importantly, BRAF-KO cells reconstituted with the BRAF-T121I mutant showed a higher growth rate than those reconstituted with BRAF-WT (Fig. 7a, b). Similarly, Transwell assay results showed that BRAF-T121I mutant expression resulted in increased cell migration and invasion compared to BRAF-WT (Fig. 7c, d). Moreover, the 3D sphere formation assay results showed that the number and size of spheres in BRAF KO cells reconstituted with the BRAF-T121I mutant were markedly increased compared to those reconstituted with BRAF-WT, suggesting that the cancer-derived BRAF mutant increased anchorage-independent cell growth (Fig. 7e, f ). Taken together, our data indicate that loss of SPOP-mediated BRAF ubiquitination promotes cell malignancy.

Discussion
Aberrant activation of the MAPK/ERK cascade is common in human cancer. BRAF mutations are frequently observed in melanoma, thyroid cancer, and others. In contrast, oncogenic BRAF mutations are uncommon in EC and PCa, with prevalence rates ranging from 2 to 5%, depending on the specific case series [23,24]. Whether specific genetic alterations drive aberrant regulation of wild-type BRAF activity in EC and PCa remains poorly understood. In the current study, we showed that the CRL3-SPOP complex mediates the non-degradative ubiquitination of BRAF, which reduces the interaction between BRAF and other MAPK/ERK pathway components, resulting in subsequent MAPK/ERK inactivation (Fig. 8). Importantly, EC-or PCa-associated SPOP mutants showed reduced capacity to ubiquitinate BRAF. Moreover, some cancer-associated BRAF mutants (T121I and S122Y) evaded SPOP-mediated regulation and were stronger activators of the MAPK/ERK cascade than wild-type BRAF. We noted that the interactions between BRAF and the MAPK/ERK pathway components were only moderately increased in BRAF-T121 reconstituted cells to BRAF-WT-reconstituted cells. It would be interesting to investigate whether this effect is more obvious in growth factor-treated conditions. Taken together, our data raise the possibility that disruption of the SPOP-BRAF regulatory axis may promote malignant transformation of cancer cells.
Proteins can be tagged with mono-ubiquitin or modified by polyUb chains that can vary in length and linkage specificity, and these variations influence how ubiquitination signals are interpreted [25]. Accumulating evidence suggests that both degradative and nondegradative ubiquitination play vital roles in modulating BRAF protein stability and function. RNF149, FBXW7, and TRAF2 have been reported as E3 ubiquitin ligases of BRAF [26][27][28]. USP28, a deubiquitinating enzyme, causes BRAF degradation by stabilizing FBXW7 [29]. In melanoma cells treated with proinflammatory cytokines, the HECT domain-containing E3 ligase ITCH catalyzes the non-degradative K27-linked ubiquitination of BRAF, which leads to PP2A recruitment, 14-3-3 dissociation, and sustained BRAF/MEK/ERK pathway activation [30]. This effect is distinct from our finding that SPOP-mediated BRAF ubiquitination inactivates BRAF. One possible explanation for this discrepancy could be that the different linkage types of polyUb chains catalyzed by CRL3-SPOP or ITCH determine the distinct fates of BRAF. Whether PolyUb chains on BRAF catalyzed by CRL3-SPOP directly hinder BRAF complex assembly or recruit other ubiquitin-binding proteins should be investigated in future studies.
The CRL3-SPOP complex catalyzes both degradative and non-degradative ubiquitination depending on the substrate. For example, CRL3-SPOP catalyzes K27-linked non-degradative ubiquitination of Geminin, which prevents DNA replication over-firing by indirectly blocking the association of Cdt1 with the MCM protein complex The p-values were calculated using the One-way ANOVA test in a, c, d, f and the Two-way ANOVA test in b. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 [31]. CRL3-SPOP catalyzed mixed K6-, K27-, and K29linked non-degradative ubiquitination of p62, which suppresses p62-dependent autophagy [32]. CRL3-SPOP catalyzes mixed K27-, K29-, K33-, K48-, and K63-linked non-degradative ubiquitination of INF2 and HIPK2 to regulate mitochondrial division and DNA damage response, respectively [19,22]. In the current study, we found that SPOP-mediated BRAF ubiquitination had no predominant ubiquitination linkage specificity, in sharp contrast to other SPOP substrates reported using Ub linkage mutants. However, the mechanisms underlying this phenomenon are currently unknown. Given that Ub linkage mutants have been artificially generated, nonphysiological effects may occur. Alternatively, we used a panel of linkage-specific Ub antibodies to demonstrate that ubiquitinated BRAF at least contains K27-, K29-, K48-, and K63-Ub linkages. Further studies are needed to thoroughly clarify the polyUb-linkage composition using mass spectrometry-based proteomics, such as the selected reaction monitoring (Ub-AQUA-SRM) method [33]. Targeted treatment with BRAF and MEK inhibition has demonstrated clinical efficacy in melanomas harboring the BRAF-V600E/K mutation, and the combination of dabrafenib and trametinib, vemurafenib, and cobimetinib represent effective therapeutic options for these patients [5]. However, vemurafenib and dabrafenib caused a paradoxical induction of MAPK/ERK pathway activation and induced proliferation via a RAS-dependent mechanism in wild-type BRAF cells [34]. AZ304, a potent dual BRAF inhibitor of both wild-type and BRAF-V600E mutant BRAF, exerts anti-tumor effects in colorectal cancer independent of BRAF genetic status [35]. Given that BRAF activity is elevated in SPOP-mutated cancers, the efficacy of AZ304 in these cancers should be investigated.

Conclusions
Our findings show that SPOP and BRAF are biochemically linked. SPOP promoted non-degradative ubiquitination of BRAF, which resulted in subsequent MAPK/ ERK inactivation. Importantly, PCa-or EC-associated SPOP mutations lead to abnormal activation of the MAPK/ERK pathway in a BRAF-dependent manner. Our data raise the possibility that disruption of the SPOP-BRAF regulatory axis may promote the malignant transformation of cancer cells (Fig. 8). Given that BRAF activity is elevated in SPOP-mutated cancers, elucidation of the SPOP-BRAF regulatory axis in further studies will be helpful in designing therapeutic strategies for SPOPmutated cancers.
Additional file 1: Figure S1. SPOP promotes non-degradative ubiquitination of BRAF (related to Figure 2). (a) Western blot of the indicated proteins in WCL from Ishikawa and KLE cells infected with lentivirus expressing SPOP-specific shRNAs or negative control. (b) Schematic of CRISPR/ Fig. 8 A model was proposed according to the findings of the present study. The schematic diagram depicts a model that SPOP regulates the MAPK/ERK pathway through promoting BRAF ubiquitination Cas9-mediated knockout of SPOP by sgRNA#1 or sgRNA#2 in Ishikawa cells. (c) Sanger sequencing confirming that the SPOP gene was edited by sgRNA#1 or sgRNA#2 in Ishikawa cells. (d) Western blot of the products of in vitro ubiquitination assays performed by incubating the reconstituted SPOP-CUL3-RBX1 E3 ligase complex with E1 and E2 enzymes, ubiquitin and GST-BRAF at 30 °C for 2 h. (e) Western blot of the products of in vivo ubiquitination assays from 293T cells transfected with the indicated plasmids. Figure S2. The SBC motif in BRAF is recognized by SPOP (related to Figure 3). (a, b) Western blot of WCL and co-IP samples of anti-FLAG antibody obtained from 293T cells transfected with the indicated plasmids. Figure S3. SPOP suppresses the activation of MAPK/ERK cascade (related to Figure 6). (a, b) SPOP-KO Ishikawa cells stably overexpressing SPOP-Y87C, or S80R mutant were serum-starved for 48 hr and then treated with EGF (100 nM) for the corresponding times. The WCLs were prepared for WB analysis. At each time point, the intensity of p-ERK1/2, p-MEK1/2, p-MSK1 was normalized to the intensity of ERK1/2, MEK1/2, MSK1 respectively and then to the value at 0 min (b). (c) Schematic of CRISPR/Cas9-mediated knockout of BRAF by sgRNA in Ishikawa cells. (d) Sanger sequencing confirming that the BRAF gene was edited by sgRNA in Ishikawa cells. (e) Western blot of the indicated proteins in WCLs from parental and BRAF-KO Ishikawa cells. All data shown are mean values ± SD(n=3). The p-values were calculated using the Two-way ANOVA test in (b). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Figure S4. The impact of SPOP-WT and EC-associated SPOP mutants on the growth of three EC cell lines. (a-c) CCK-8 assays in KLE (a), SPEC-2 (b), and Ishikawa (c) cells stably overexpressing EV, SPOP-WT or EC-associated SPOP mutants. Data are shown as the means ± SD (n=4). The p-values were calculated using the Two-way ANOVA test in (a-c). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. (d) Western blot of the indicated proteins in WCL from KLE, SPEC-2, and Ishikawa cells stably overexpressing EV, SPOP-WT, or EC-associated SPOP mutants. Table S1. Sequence information. Table S2. Antibody and recombinant protein information. Table S3. Cell cultures, chemicals and Kit.